Method for Automatic Determination of Sample

ABSTRACT

A method of determining a kind of a sample, in a method for analyzing a substance by the steps of supplying the sample to be analyzed to a reaction system by a supplying means comprising a transparent region composed of a transparent material, reacting a reagent for detecting the substance with the sample in the reaction system, and analyzing a signal derived from a product obtained by the reaction, characterized by irradiating the transparent region with light in the supplying step, and analyzing an optical intensity of the light.

TECHNICAL FIELD

The present invention relates to a method for an automatic determinationof a sample type, in a method for analyzing the sample, i.e., a methodfor analyzing a specific component contained in the sample such as bodyfluids, particularly blood.

BACKGROUND ART

It is clinically important to measure a specific component in blood, forexample, antigens, antibodies, proteins, or endocrines. A serum orplasma is commonly used as a blood sample and, in this case, a serum orplasma is generally separated from whole blood as rapidly as possible toavoid hemolysis. This is because, when blood cells are contained in asample or hemolysis occurs, for example, in an immunoassay, there is apossibility of the occurrence of the effects of hemolysis on an opticalsystem, an inhibition of an immunoreaction by internal components ofblood cells, an aggregation of insoluble carriers used as a solid phasecaused by a membrane component of blood cells, or an interference byadsorption or the like. Therefore, it is a conventional procedure ingeneral clinical laboratory tests to remove blood cells from collectedwhole blood by centrifugation and to use the resulting serum or plasmaas a sample to be tested.

However, since a dedicated device such as a centrifuge is necessary forthe removal of blood cells, and the procedure needs time and effort, itis preferable to use whole blood as a sample without pretreatment, for ageneral practitioner not having such a device or in an unrespitedemergency test, and various methods have been proposed.

For example, Japanese Unexamined Patent Publication (Kokai) No. 10-48214(patent reference 1) discloses a method for using completely hemolyzedwhole blood by sonicating whole blood or mixing whole blood with ahypotonic solution. Japanese Unexamined Patent Publication (Kokai) No.6-265554 (patent reference 2) discloses a method of analyzing abiochemical component of blood, comprising the steps of determiningwhether or not a sample contains blood cells; when the result that thesample contains blood cells is obtained, determining whether or not onlyone or more measurable items which can be analyzed by using a samplecontaining blood cells are selected; and when the result that onlymeasurable item(s) which can be analyzed by using a sample containingblood cells are selected is obtained, stirring the sample and measuringthe stirred sample.

However, the method for completely hemolyzing whole blood disclosed inthe above patent reference 1 has several problems, for example, variousstates of hemolysis. Further, interferences which flow from the insideof blood cells to a reaction system, such as hemoglobin or substancesderived from the cell nucleus, sometimes seriously affect themeasurement by causing a nonspecific reaction or, particularly in animmunological assay, an interfering of immunoreaction.

With respect to the method of analyzing a biochemical component of blooddisclosed in the above patent reference 2, a method of determining thekind of the sample (i.e., whether or not the sample contains bloodcells) is not fully disclosed. The patent reference 2 only disclosesthat a means for determining the kind of a sample, such as atransmission optical sensor, may be located over cuvettes encapsulatinga reagent, a sample, a diluent, or the like. Further, a concreteprocedure and criterion for the determination are not disclosed in thepatent reference 2, except for the disclosure that, when a samplecontains blood cells, a hematocrit compensation should be carried out bystirring the sample.

[patent reference 1] Japanese Unexamined Patent Publication (Kokai) No.10-48214[patent reference 2] Japanese Unexamined Patent Publication (Kokai) No.6-265554

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method capable ofautomatically determining the kind of a sample, in a general-purposeautomatic analyzer for not only a serum or plasma but also whole blood,without a step in which a measurer inputs or sets the type of the sampleinto the analyzer before the measurement. Another object is to provide amethod capable of detecting a failure to apply a sample into theanalyzer, simultaneously with the determination of the type of thesample. Still another object is to provide a method capable ofsimultaneously detecting a failure to mount a dispensing tip, in anautomatic analyzer in which dispensing tips are mounted.

Means for Solving the Problems

These objects may be solved by the present invention, i.e., a method ofdetermining a kind of a sample (preferably the presence or absence of asample in a supplying means and/or a kind of a sample), in a method foranalyzing a substance by the steps of supplying the sample suspected ofcontaining the substance to be analyzed to a reaction system by asupplying means comprising a transparent region composed of atransparent material, reacting a reagent for detecting the substancewith the sample in the reaction system, and analyzing a signal derivedfrom a product obtained by the reaction, characterized by irradiatingthe transparent region with light in the supplying step, and analyzingan optical intensity of the light.

According to a preferred embodiment of the method of the presentinvention, the supplying means is a dispensing means in which a tip canbe mounted (preferably, removably mounted) and liquid can be aspiratedand poured out by means of the tip. That is, the present inventionrelates to a method of determining a kind of a sample (preferably thepresence or absence of a sample in a tip and/or a kind of a sample, morepreferably the mounting or detaching of a tip, the presence or absenceof a sample in a tip, and/or a kind of a sample), in a method foranalyzing a substance by the steps of supplying (preferably dispensing)the sample suspected of containing the substance to be analyzed to areaction system by a dispensing means in which a tip is mounted andliquid can be aspirated and poured out by means of the tip, reacting areagent for detecting the substance with the sample in the reactionsystem, and analyzing a signal derived from a product obtained by thereaction, characterized by irradiating a sample-holding portion of thetip with light in the supplying step (i.e., when the sample is aspiratedinto the tip), and analyzing an optical intensity of the light.

According to another preferred embodiment of the method of the presentinvention, the supplying means is a tube or a channel. That is, thepresent invention relates to a method of determining a kind of a sample(preferably the presence or absence of a sample in a supplying meansand/or a kind of a sample), in a method for analyzing a substance by thesteps of supplying the sample suspected of containing the substance tobe analyzed to a reaction system by a tube or a channel comprising atransparent region composed of a transparent material, reacting areagent for detecting the substance with the sample in the reactionsystem, and analyzing a signal derived from a product obtained by thereaction, characterized by irradiating the transparent region with lightin the supplying step (i.e., when the sample passes through the tube orthe channel), and analyzing an optical intensity of the light.

According to still another preferred embodiment of the method of thepresent invention, the sample is whole blood, a serum, or plasma.

The present invention relates to an apparatus for analyzing a substanceby the steps of supplying a sample suspected of containing the substanceto be analyzed to a reaction system by a supplying means, reacting areagent for detecting the substance with the sample in the reactionsystem, and analyzing a signal derived from a product obtained by thereaction, characterized by comprising

(a) a supplying means comprising a transparent region composed of atransparent material;(b) an irradiating means capable of irradiating the transparent regionwith light in the supplying step;(c) an optically analyzing means capable of analyzing a change in anoptical intensity of the light; and(d) a means for determining a kind of the sample by the opticalintensity.

The determining means may comprise, for example,

a means for storing a threshold value obtained from a previouslymeasured value,a means for comparing a secondary measured value with the storedthreshold value,a means for indicating a subsequent procedure (or a warning) accordingto the comparison result, anda means for outputting the comparison result (a warning means).

According to a preferred embodiment of the apparatus of the presentinvention, the supplying means is a dispensing means in which a tip canbe mounted (preferably, removably mounted) and liquid can be aspiratedand poured out by means of the tip. That is, the present inventionrelates to an apparatus for analyzing a substance by the steps ofsupplying (preferably dispensing) a sample suspected of containing thesubstance to be analyzed to a reaction system by a dispensing means inwhich a tip is mounted and liquid can be aspirated and poured out bymeans of the tip, reacting a reagent for detecting the substance withthe sample in the reaction system, and analyzing a signal derived from aproduct obtained by the reaction, characterized by comprising

(a) a dispensing means in which a tip can be mounted and liquid can beaspirated and poured out by means of the tip;(b) an irradiating means capable of irradiating a sample-holding portionof the tip with light in the supplying step (i.e., when the sample isaspirated into the tip);(c) an optically analyzing means capable of analyzing a change in anoptical intensity of the light; and(d) a means for determining a kind of the sample (preferably thepresence or absence of a sample in a tip and/or a kind of a sample, morepreferably the mounting or detaching of a tip, the presence or absenceof a sample in a tip, and/or a kind of a sample) by the opticalintensity.

According to a preferred embodiment of the apparatus of the presentinvention, the supplying means is a tube or a channel. That is, thepresent invention relates to an apparatus for analyzing a substance bythe steps of supplying a sample suspected of containing the substance tobe analyzed to a reaction system by a tube or a channel comprising atransparent region composed of a transparent material, reacting areagent for detecting the substance with the sample in the reactionsystem, and analyzing a signal derived from a product obtained by thereaction, characterized by comprising

(a) a tube or a channel comprising a transparent region composed of atransparent material;(b) an irradiating means capable of irradiating the transparent regionwith light in the supplying step;(c) an optically analyzing means capable of analyzing a change in anoptical intensity of the light; and(d) a means for determining a kind of the sample (preferably thepresence or absence of a sample in the tube or the channel and/or a kindof the sample) by the optical intensity.

According to still another preferred embodiment of the apparatus of thepresent invention, the sample is whole blood, a serum, or plasma.

Effects of the Invention

According to the present invention, the kind of a sample can beautomatically determined, and a step in which a measurer inputs or setsthe kind of the sample into an analyzer before the measurement can beskipped. Therefore, the present invention is useful, particularly, for ageneral operator not having a dedicated device such as a centrifuge orfor an unrespited emergency test. Further, according to the presentinvention, a failure to apply a sample into the analyzer can bedetected, as well as the determination of the type of the sample.Furthermore, according to the present invention, a failure to mount adispensing tip can be simultaneously detected in an automatic analyzerin which dispensing tips are mounted.

BRIEF DESCRIPTION OF THE DRAWINGS

[FIG. 1]

FIG. 1 is a front view (A) and a side view (B) which schematicallyillustrate an embodiment of an automatic analyzer to which thedetermining method of the present invention may be applied.

[FIG. 2]

FIG. 2 schematically illustrates procedures of an embodiment of anautomatic analyzing method to which the determining method of thepresent invention may be applied.

[FIG. 3]

FIG. 3 schematically illustrates an embodiment of an optical analyzingsystem which may be used in the present invention.

[FIG. 4]

FIG. 4 is an enlarged partial front view (A) and an enlarged partialside view (B) which schematically illustrate a state in which anembodiment of an optical analyzing system which may be used in thepresent invention is incorporated into an automatic analyzer.

EXPLANATIONS OF REFERENCE SIGNS IN DRAWINGS

1□□□automatic analyzer; 2□□□measuring table; 3□□□cartridge; 4□□□tip;5□□□nozzle;11□□□light emitting diode; 12□□□photodiode;13□□□operational amplifier for current-voltage conversion;14□□□converter.

BEST MODE FOR CARRYING OUT THE INVENTION

The determining method of the present invention can be applied to amethod for an automatic analysis of a sample, such as body fluids,particularly blood, so long as the automatic analyzing method comprisesa step of supplying the sample to a reaction system. As the automaticanalyzing method, there may be mentioned, for example, an automaticanalyzing method comprising a step of supplying (for example,dispensing) a sample to a reaction system by a dispensing means in whicha tip can be mounted and liquid can be aspirated and poured out by meansof the tip, or an automatic analyzing method comprising a step ofsupplying a sample to a reaction system by a transfer means (forexample, a tube or a channel) capable of aspirating the sample from oneend thereof and transferring the sample from the other end thereof.Further, the analyzing apparatus of the present invention can be appliedto an automatic analyzer equipped with a means for supplying a sample toa reaction system. As the automatic analyzer, there may be mentioned,for example, an analyzer equipped with a dispensing means in which a tipcan be mounted and liquid can be aspirated and poured out by means ofthe tip, or an analyzer equipped with a transfer means (for example, atube or a channel) capable of aspirating the sample from one end thereofand transferring the sample from the other end thereof.

An embodiment of an automatic analyzing method and an automatic analyzerto which the determining method of the present invention can be appliedwill be explained with reference to FIGS. 1 and 2.

The automatic analyzer 1 shown in FIG. 1 comprises a measuring table 2on which one or more cartridges 3 having plural wells for samples and/orreagents for detection can be placed. A line of plural nozzles 5, inwhich tips 4 can be mounted and liquid can be aspirated and poured outby means of the tips 4, are placed over the measuring table. The nozzles5 can be moved up and down in the vertical direction. When the bottomend of the tip 4 is located at the lowest position, an aspiration ofliquid from wells of the cartridge 3, pouring of liquid to wellsthereof, mixing of liquid in the wells by continuously repeatingaspiration and injection, or the like can be carried out. Further, wellsof interest can be placed directly below the nozzles by moving themeasuring table 2 in the horizontal direction. If desired, one or moremagnets (not shown in FIG. 1) capable of being brought into contact withthe outer sidewalls of tips 4 may be positioned. In this case, magneticparticles as a reagent for detection, such as magnetic particles coatedwith an antibody specific to a compound to be analyzed, may be usedtogether with the magnets to carry out a B/F separation in the tips.

As the means for supplying a sample to a reaction system, anaspiration/injection means in which a tip portion is integrated with anozzle portion, a transfer means capable of aspirating a sample from oneend thereof and transferring the sample from the other end thereof, orthe like may be used, instead of the nozzles in which tips can bemounted as shown in FIG. 1. As the transfer means, there may bementioned, for example, a tube such as a flexible tube or a capillarytube, or a channel. Hereinafter, the present invention will furtherexplained with reference to embodiments equipped with the dispensingmeans in which a tip can be mounted and liquid can be aspirated andpoured out by means of the tip, but is by no means limited to theseembodiments.

An embodiment of an automatic analyzing method using the automaticanalyzer shown in FIG. 1 is shown in FIG. 2. In the automatic analyzingmethod shown in FIG. 2, magnetic particles coated with the firstantibody, the second antibody labeled with an enzyme, and a luminescencesubstrate are used as reagent for detection, together with magnetscapable of being brought into contact with the outer walls of tips, tocarry out a B/F separation in the tips.

In the first step [(a) first reaction], a predetermined amount of asample is added, by means of a tip, to the first well to which apredetermined amount of antibody-coated magnetic particles is previouslydispensed, the sample and the antibody-coated magnetic particles areproperly mixed by pipetting, and the mixture is incubated. After theantigen-antibody reaction has proceeded sufficiently, the reactionliquid is aspirated into the tip, and the reaction liquid is poured outwhile capturing the magnetic particles on the inner wall of the tip witha magnet.

In the second step [(b) washing], the tip is inserted in the second wellto which a predetermined amount of a washing solution is previouslydispensed, and the magnetic particles captured with the magnet arewashed by pipetting of the washing solution.

In the third step [(c) second reaction], the tip is inserted in thethird well to which a predetermined amount of an enzyme-labeled antibodysolution is previously dispensed, the magnetic particles and thesolution are properly mixed by pipetting of the labeled antibodysolution, and the mixture is incubated. After the antigen-antibodyreaction has proceeded sufficiently, the reaction liquid is aspiratedinto the tip, and the reaction liquid is poured out while capturing themagnetic particles on the inner wall of the tip with the magnet.

In the fourth step [(d) washing], the tip is inserted in the fourth wellto which a predetermined amount of a washing solution is previouslydispensed, and the magnetic particles captured with the magnet arewashed by pipetting of the washing solution.

In the fifth step [(e) luminescence reaction], the tip is inserted inthe fifth well to which a predetermined amount of a luminescencesubstrate solution is previously dispensed, and a luminescence reactionis carried out by pipetting of the substrate solution. After thereaction is carried out for a predetermined time, an amount ofluminescence may be measured to determine an amount or concentration ofa substance to be analyzed.

As a sample to be analyzed used in the present invention, there may bementioned, for example, body fluids, particularly blood, such as wholeblood, a serum, or plasma.

A substance to be analyzed contained in the sample is not particularlylimited, so long as a substance which specifically binds with theanalyte to form a reaction product can be selected. As a combination ofthe analyte and a substance specific thereto, there may be mentioned,for example, an antigen and an antibody, an antibody and an antigen, aprotein and a ligand, or a sugar chain and a lectin, preferably anantigen and antibody, or an antibody and an antigen. The term “tospecifically bind” used herein means to form a reaction product bybiochemically and specifically binding with a subject. As the substanceto be analyzed, there may be mentioned, for example, hepatitis B virussurface antigen (HBsAg), hepatitis C virus (HCV) antibody and antigen,human immunodeficiency virus (HIV) antibody, human T-cell leukemiavirus-1 (HTLV-1) antibody, or Treponema pallidum (TP) antibody. Further,myocardial markers [for example, creatine kinase MB (CKMB), myoglobin,or troponin], hormones, serum proteins, or the like may be used.

A reaction system for measuring a sample is not particularly limited.For example, an immunoassay based on an antigen-antibody reaction may bepreferably used.

The determination in the present invention is based on an opticaltechnique. In the present invention, a sample is aspirated into a tip,and a portion in which the sample is held is irradiated with light. Theoptical intensity may be analyzed to determine whether or not the tip ismounted, whether or not the sample exists in the tip, and/or the kind ofthe sample.

More particularly, with respect to the tip for aspirating a sample, aportion in which the sample is held is irradiated with light, preferablybefore the aspiration and after the aspiration, and an optical changein, for example, transmission, reflection, or scattering may be detectedwith a well-known device such as a photodetector to determine whether ornot the tip is mounted, whether or not the sample exists in the tip,and/or the kind of the sample.

When a tube or a channel comprising a transparent region composed of atransparent material is used as the supplying means, the transparentregion is irradiated with light, and an optical intensity, which variesaccording to the presence or absence of the sample and/or the kind ofthe sample transferred in the tube or the channel, may be analyzed todetermine the presence or absence of the sample and/or the kind of thesample.

As shown in the experimental data of Examples 1 and 2 described below,an amount of transmitted light when a tip is not mounted (hereinafterreferred to as the unmounted-tip case) is larger than that when a tip ismounted and no sample is aspirated into the tip (hereinafter referred toas the mounted-tip case), and thus, it is possible to determine whetheror not a tip is mounted by measuring an amount of transmitted light.

Further, since little light is transmitted when whole blood is aspiratedinto a tip (hereinafter referred to as whole-blood-holding case), anamount of transmitted light in the whole-blood-holding case becomessmaller than that in the mounted-tip case. In contrast, an amount oftransmitted light when plasma or a serum is aspirated into a tip(hereinafter referred to as plasma-or-serum-holding case) becomes largerthan that in the mounted-tip case, due to the lens effect caused by atip, but does not exceed that in the unmounted-tip case.

These cases can be arranged in order of the decreasing amount oftransmitted light as follows:

unmounted-tip case>plasma-or-serum-holding case>mounted-tipcase>whole-blood-holding case.

Therefore, it is possible to determine whether or not a tip is mounted,whether or not a sample exists in a tip, and/or the kind of a sample, byusing an amount of transmitted light as an index. In the unmounted-tipcase, an inadequate operation, such as a failure in preparing tips, afailure in mounting a tip, or the like, can be detected. In themounted-tip case, an inadequate operation, such as a failure inpreparing samples, a failure in setting one or more samples, or thelike, can be detected. Each threshold may vary according to conditions,such as the kind of an optical analyzing system, properties (forexample, materials, the quality of materials, shape, or size) of a tip,or the like, and thus, is not particularly limited. Those skilled in theart can easily determine each threshold without undue experimentation bycarrying out a pilot test, for example, by measuring amounts oftransmitted light in these cases in accordance with the proceduresdescribed in Examples 1 to 2.

With respect to three cases that a sample is (1) whole blood, (2) plasmaor a serum, and (3) an inadequate operation (for example, a failure insetting one or more samples), an embodiment of a logic for determinationwill be explained below.

First, a pilot test using plural whole blood and plasma and/or sera iscarried out to measure amounts of transmitted light when a sample isaspirated into a tip (i.e., an amount of transmitted light in thewhole-blood-holding case, and an amount of transmitted light in theplasma-or-serum-holding case). Further, an amount of transmitted light(To) in the mounted-tip case (i.e., when no sample is aspirated into atip) is measured. As described above, the order of the decreasing amountof transmitted light is as follows:

-   -   plasma-or-serum-holding case>mounted-tip        case>whole-blood-holding case.        On the basis of these measurement values, a threshold between        the plasma-or-serum-holding case and the mounted-tip case        (hereinafter referred to as threshold a) and a threshold between        the mounted-tip case and the whole-blood-holding case        (hereinafter referred to as threshold b) are determined in        advance.

Next, to determine a unknown sample, the tip is irradiated with light tomeasure an amount of transmitted light (T). When the amount oftransmitted light T of the unknown sample is larger than the thresholda, it is possible to determine that this is the plasma-or-serum-holdingcase, i.e., that the unknown sample is plasma or a serum. When theamount of transmitted light T of the unknown sample is smaller than thethreshold b, it is possible to determine that this is theplasma-or-serum-holding case, i.e., that the unknown sample is plasma ora serum. When the amount of transmitted light T of the unknown sample isbetween the threshold a and the threshold b, it is possible to determinethat there is an inadequate operation.

The above logic for determination (hereinafter sometimes referred to asthe one-step method) which may be used in the present inventioncomprises, for example, the steps of:

(A) comparing an optical intensity (for example, an amount oftransmitted light) measured in a supplying step (for example, when asample to be determined is aspirated into a supplying means such as atip) with the previously determined threshold a (i.e., the thresholdbetween an optical intensity when plasma or a serum is aspirated intothe supplying means and an optical intensity when no sample is aspiratedinto the supplying means) and the previously determined threshold b(i.e., the threshold between an optical intensity when no sample isaspirated into the supplying means and an optical intensity when wholeblood is aspirated into the supplying means); and(B) determining that when the optical intensity of the sample is higherthan the threshold a, the sample is plasma or a serum; when the opticalintensity thereof is lower than the threshold b, the sample is wholeblood; and when the optical intensity thereof is between the threshold aand the threshold b, no sample is aspirated into the supplying means(for example, an inadequate operation).

According to the present invention, even if a sample is a hemolyticsample or a usual chyle sample (i.e., a whitish and opaque sample havinga high lipid content), plasma or a serum can be clearly discriminatedfrom whole blood, as shown in the experimental data described in Example2. In this connection, when a chyle plasma or serum sample having anextremely high lipid content is used, it is sometimes difficult todiscriminate the sample from whole blood. For example, when a chylesample having an extremely high lipid content is used, an amount oftransmitted light is sometimes a value between the threshold a and thethreshold b (i.e., judged as an inadequate operation) or smaller thanthe threshold b (i.e., judged as whole blood) in the above-mentionedlogic for determination. In such a case, when samples to be analyzedinclude (or are suspected of including) one or more chyle samples havingan extremely high lipid content, a chyle plasma or serum sample can bediscriminated from whole blood by diluting the sample(s) to, forexample, 1.2-fold to 10-fold, preferably 1.5-fold to 5-fold, moreparticularly 2-fold, and measuring again an amount of transmitted lightof the diluted sample(s). That is, when the sample is whole blood, thereis little change between the values measured before the dilution andafter the dilution, and when the sample is chyle plasma or serum, thevalue measured after the dilution is increased, and thus, a chyle plasmaor serum sample can be discriminated from whole blood.

More particularly, when the amount of transmitted light T of an unknownsample (in undiluted form) is smaller than the threshold b (a usualsample is judged as whole blood) in the above-mentioned logic fordetermination, the unknown sample is diluted, and an amount oftransmitted light of the diluted sample (hereinafter referred to as theamount of transmitted light T′) is measured again under the sameconditions. In this case, when the unknown sample is a chyle plasma orserum sample, the amount of transmitted light measured after thedilution is increased. In contrast, when the unknown sample is wholeblood, there is little change between the values measured before thedilution and after the dilution. Therefore, such an unknown sample canbe discriminated by previously determining a threshold c, with respectto the difference (T′-T) between the amount of transmitted light afterthe dilution (T′) and that before the dilution (T). That is, when thedifference “T′-T” is larger than the threshold c, the unknown sample maybe judged as a chyle plasma or serum sample, and when the difference“T′-T” is not more than the threshold c, the unknown sample may bejudged as whole blood.

Similarly, when the amount of transmitted light T of an unknown sample(in undiluted form) is between the threshold a and the threshold b (ausual sample is judged as that when no sample is aspirated into a tip)in the above-mentioned logic for determination, the unknown sample isdiluted, and the amount of transmitted light T′ is measured again underthe same conditions. When the unknown sample is a chyle plasma or serumsample, the amount of transmitted light measured after the dilution isincreased. In contrast, when no sample is aspirated into a tip, there islittle change between the values measured before the dilution and afterthe dilution. Therefore, such an unknown sample can be discriminated bypreviously determining a threshold d, with respect to the difference(T′-T) between the amount of transmitted light after the dilution (T′)and that before the dilution (T). That is, when the difference “T′-T” islarger than the threshold d, the unknown sample may be judged as a chyleplasma or serum sample, and when the difference “T′-T” is not more thanthe threshold d, it may be judged that no sample is aspirated into atip.

As above, another logic for determination (hereinafter sometimesreferred to as the two-step method) which may be used in the presentinvention comprises, for example, the steps of:

(A) comparing an optical intensity (for example, an amount oftransmitted light) T measured in a supplying step (for example, when asample to be determined is aspirated into a supplying means such as atip) with the previously determined threshold a (i.e., the thresholdbetween an optical intensity when plasma or a serum is aspirated intothe supplying means and an optical intensity when no sample is aspiratedinto the supplying means) and the previously determined threshold b(i.e., the threshold between an optical intensity when no sample isaspirated into the supplying means and an optical intensity when wholeblood is aspirated into the supplying means);(B′) determining that, when the optical intensity of the sample ishigher than the threshold a, the sample is judged as plasma or a serum;when the optical intensity thereof is lower than the threshold b, thefollowing step (C) is carried out; and when the optical intensitythereof is between the threshold a and the threshold b, the followingstep (D) is carried out;(C) diluting the sample, and comparing the difference (T′-T) between theoptical intensity T′ measured under the same conditions and the opticalintensity T, with the previously determined threshold c (i.e., athreshold between the difference “T′-T” when a sample is a chyle plasmaor serum sample and the difference “T′-T” when a sample is whole blood),to determine that, when the “T′-T” is larger than the threshold c, thesample is judged as a chyle plasma or serum sample, and when thedifference “T′-T” is not more than the threshold c, the unknown sampleis judged as whole blood; and(D) diluting the sample, and comparing the difference (T′-T) between theoptical intensity T′ measured under the same conditions and the opticalintensity T, with the previously determined threshold d (i.e., athreshold between the difference “T′-T” when a sample is a chyle plasmaor serum sample and the difference “T′-T” when no sample is aspiratedinto the supplying means), to determine that, when the “T′-T” is largerthan the threshold d, the sample is judged as a chyle plasma or serumsample, and when the difference “T′-T” is not more than the threshold d,it is judged that no sample is aspirated into the supplying means (forexample, an inadequate operation).

The logic for determination in the two-step method is shown in Table 1.

TABLE 1 Step B′$\frac{T < b}{{to}\mspace{14mu} {Step}\mspace{14mu} C}$$\frac{b \leq T \leq a}{{to}\mspace{14mu} {Step}\mspace{14mu} D}$$\frac{a < T}{P/S}$ Step C $\frac{{\Delta T} \leq c}{WB}$$\frac{c < {\Delta T}}{P/S}$ Step D $\frac{{\Delta T} \leq d}{IO}$$\frac{d < {\Delta T}}{P/S}$ WB: whole blood P/S: plasma or a serum IO:inadequate operation

In the present invention, the one-step method or the two-step method maybe appropriately selected and carried out according to the state ofsamples (a set of samples) to be analyzed. For example, when the samplesto be analyzed include no chyle sample having an extremely high lipidcontent, the one-step method is preferable. A chyle sample can beidentified by, for example, a visual check. According to the one-stepmethod, a convenient and rapid analysis can be carried out, because thejudgment is made in one step. When the samples to be analyzed include(or are suspected of including) one or more chyle samples having anextremely high lipid content, the two-step method is preferable.According to the two-step method, a more accurate analysis can becarried out, and a visual check of samples is not necessary. The effectof chyle (lipid content in samples) was examined using intrafat (20%,Takeda Chemical Industries, Ltd.) and, as a result, the type of sampleshaving a lipid content of 300 mg/dL or less could be determined by theone-step method of the present invention. Further, with respect tosamples having a lipid content of more than 300 mg/dL, it was confirmedthat the type of samples having a lipid content of 1500 mg/dL or lesscould be determined (particularly, discriminated from whole blood) bythe two-step method (2-fold dilution).

An embodiment of an optical analyzing system (transmission type) whichmay be used in the present invention is shown in FIGS. 3 and 4.

As shown in FIG. 3, a light emitting diode (LED) 11 and a photodiode(PD) 12 are located opposite to each other and spaced apart, so that atip 4 can be placed therebetween. As the tip, a material through whichlight can be transmitted may be used, and there may be mentioned, forexample, glass or transparent plastics, such as polyethylene,polystyrene, polycarbonate, polyacrylic plastics, or polypropylene. Theinner diameter, wall thickness, material, or the like of the tip, andthe light intensity, optical path length, or the like of the light maybe appropriately selected to optimize the optical system. For example,when polypropylene tips are used, the portion to be irradiated withlight has an outer diameter of preferably 2 to 10 mm, more preferably 3to 6 mm, an inner diameter of preferably 1 to 8 mm, more preferably 2 to4 mm, and a wall thickness of preferably 0.2 to 2 mm, more preferably0.5 to 1 mm. In this connection, the present invention is not limited tothese values.

The wavelength of the photodiode is not limited, so long as at least theunmounted-tip case, the plasma-or-serum-holding case, the mounted-tipcase, and the whole-blood-holding case are optically discriminated fromeach other. The wavelength of light emitted from a commonly usedphotodiode includes an ultraviolet region, a visible region, and aninfrared region, and an appropriate wavelength may be selectedtherefrom. For example, a visible region of 380 to 780 nm may bepreferably used, more preferably 400 to 700 nm, most preferably 470 to635 nm. In this connection, the present invention is not limited tothese wavelength ranges.

With respect to the irradiation angle of light, it is preferable toirradiate the tip with light at a right angle to the tip. When theirradiation angle is not a right angle, the present invention can becarried out by using a means for compensating a refractive index causedwhen the light is transmitted through the tip and the sample.

As a means for detecting the transmitted light, a well-known detectingmeans may be used, with appropriate modifications if desired. Forexample, the light output from the light source (LED) 11 is transmittedthrough the tip, the amounts of light (current value) detected with thephotodetector (photodiode) 12 are current-voltage converted by theoperational amplifier 13, the analog values are digitalized with the ADconverter 14, and the digital values are processed with software.

In this procedure, digital values or levels according to the types ofsamples and the presence or absence of the tip can be input as thethresholds in advance.

In the present invention, as an optical analyzing system other than thatshown in FIGS. 3 and 4, for example, an image processing system using aCCD camera may be used.

In the image processing system using a CCD camera, the detected lightcan be captured as color information by passing the light through an RGBprimary-colors filter, and the sample can be determined by color. Whenthe RGB primary-colors filter is not used, it can be determined fromgradations in monochrome whether or not the light is transmitted.

EXAMPLES

The present invention now will be further illustrated by, but is by nomeans limited to, the following Examples.

Example 1 Determination of Whole Blood and Plasma by Various Wavelengths

In this example, the system shown in FIGS. 3 and 4 for analyzingtransmitted light was used to determine whole blood and plasma.

As a light emitting diode (LED), three types of LEDs, i.e., an LEDhaving a peak wavelength of 635 nm (GL3HD44; Stanley), an LED having apeak wavelength of 573 nm (NSPY800AS; NICHIA), and an LED having a peakwavelength of 470 nm (NSPB500S; NICHIA), were used. As a photodiode(PD), a PD having a sensitivity wavelength range of 320 to 1100 nm(S6775; Hamamatsu Photonics) was used.

As a tip, polypropylene tips having a portion (outer diameter=3.6 mm,inner diameter=2.2 mm, and wall thickness=0.7 mm) irradiated with lightwere used.

As a sample aspirated into the tip, water (purified water), plasma, andwhole blood were used. With respect to the cases that the tip was notmounted, that the tip was mounted, and that one of the samples wasaspirated into the tip, an amount of transmitted light was measuredusing the system shown in FIGS. 3 and 4. The results are shown in Table2. The unit used in Table 2 is output voltage (V).

As shown in Table 2, a detected value of light was highest when the tipwas not mounted (i.e., control), and the detected value of light waslowered by mounting the tip. Whereas the detected value of light wasfurther lowered when whole blood was aspirated into the tip, thedetected value of light was increased due to the lens effect caused bythe tip when water or plasma was aspirated into the tip, in comparisonwith the case of the tip alone.

It was clarified from these results that whole blood can bediscriminated from plasma by measuring the detected value of light.Further, the difference between the value obtained in the case of thetip alone and that of the tip and plasma was enough to discriminate onefrom the other, and thus, the cases of no tip, tip alone, plasma, andwhole blood can be discriminated automatically. In this connection,similar results were obtained with respect to a serum.

TABLE 2 470 nm 575 nm 635 nm Control 3.52 3.49 3.29 Tip alone 0.94 1.171.26 Tip and water 1.73 2.13 2.12 Tip and plasma 1.39 1.99 2.04 Tip andwhole blood 0.17 0.12 0.69

Example 2 Determination of Various Samples

In this example, the procedures described in Example 1 were repeated,except that an LED having a peak wavelength of 590 nm (EFY3863; Stanley)was used as an LED for a light source.

Plasma, plasma supplemented with commercially available interferingsubstances (purchased from Sysmex Corporation), whole blood, and waterwere used as samples. The interfering substances used and finalconcentrations (or turbidity) thereof are as follows:

□bilirubin (concentration=25 mg/dL, 50 mg/dL, and 75 mg/dL)□hemoglobin (concentration=500 mg/dL, 750 mg/dL, 1000 mg/dL, and 1500mg/dL)□chyle (formazin turbidity) (concentration=1500 degree, 3000 degree, and4500 degree)

With respect to the cases that the tip was not mounted, that the tip wasmounted, and that one of the samples was aspirated into the tip, anamount of transmitted light was measured in accordance with theprocedure described in Example 1. Each sample was measured 18 times, andthe averages thereof are shown in Table 3. The unit used in Table 3 isoutput voltage (V).

As shown in Table 3, a detected value of light was highest when the tipwas not mounted, and the detected value of light was lowest when wholeblood was aspirated in the tip. Detected values of light when liquid(plasma, plasma supplemented with bilirubin, plasma supplemented withhemoglobin, plasma supplemented with chyle, or water) was aspirated werebetween that in the control and that in the whole-blood-holding case.These results indicate that whole blood can be discriminated from plasmaby measuring the detected value of light. Further, it was clarified fromthese results that, even if plasma is colored with hemolysis or thelike, or plasma is an opaque plasma having a high lipid content, wholeblood can be clearly discriminated from such plasma.

TABLE 3 Samples to be measured Output voltage control (no tip) 2.99 tipalone 0.88 water 2.10 plasma 1.88 with bilirubin, 25 mg/dL 1.92 withbilirubin, 50 mg/dL 1.94 with bilirubin, 75 mg/dL 1.93 with hemoglobin,500 mg/dL 1.89 with hemoglobin, 750 mg/dL 1.92 with hemoglobin, 1000mg/dL 1.83 with hemoglobin, 1500 mg/dL 1.82 with chyle (turbidity), 1500degree (FTU) 1.84 with chyle (turbidity), 3000 degree (FTU) 1.87 withchyle (turbidity), 4500 degree (FTU) 1.74 whole blood 0.66

Example 3 Discrimination Between Chyle Sample and Whole Blood

The following procedure was carried out in accordance with theprocedures described in Example 2.

Intrafat (20%, Takeda Chemical Industries, Ltd.) was used to preparesamples having concentrations (lipid) shown in Table 4. These samplesand whole blood were used as samples.

With respect to the cases that one of samples (the above-mentionedsamples and the double-diluted samples thereof) was aspirated into thetip, an amount of transmitted light was measured in accordance with theprocedure described in Example 1. Each sample was measured 18 times, andthe averages thereof are shown in Table 4. The unit used in Table 4 isoutput voltage (V).

As shown in Table 4, it was clarified that samples suspected of beingmisidentified as whole blood due to the effect of chyle (300 mg/dL ormore) can be clearly discriminated from whole blood by the two-stepmethod.

TABLE 4 One-step method Two-step method Samples Output voltage Outputvoltage  200 mg/dL 1.64 1.92  300 mg/dL 1.23 1.89  350 mg/dL 1.01 1.88 500 mg/dL 0.79 1.80 1000 mg/dL 0.73 1.74 1500 mg/dL 0.70 1.71 Wholeblood 0.67 0.65

INDUSTRIAL APPLICABILITY

The present invention may be applied to an automatic analysis of samplessuch as body fluids.

Although the present invention has been described with reference tospecific embodiments, various changes and modifications obvious to thoseskilled in the art are possible without departing from the scope of theappended claims.

1. A method of determining a kind of a sample, in a method for analyzinga substance by the steps of supplying the sample to be analyzed to areaction system by a supplying means comprising a transparent regioncomposed of a transparent material, reacting a reagent for detecting thesubstance with the sample in the reaction system, and analyzing a signalderived from a product obtained by the reaction, characterized byirradiating the transparent region with light in the supplying step, andanalyzing an optical intensity of the light.
 2. The method according toclaim 1, wherein the supplying means is a dispensing means in which apipette tip can be mounted and liquid can be aspirated and poured out bymeans of the tip.
 3. The method according to claim 1, wherein thesupplying means is a tube or a channel.
 4. The method according to claim1, wherein the sample is whole blood, a serum, or plasma.
 5. Anapparatus for analyzing a substance by the steps of supplying a sampleto be analyzed to a reaction system by a supplying means, reacting areagent for detecting the substance with the sample in the reactionsystem, and analyzing a signal derived from a product obtained by thereaction, characterized by comprising (a) a supplying means comprising atransparent region composed of a transparent material; (b) anirradiating means capable of irradiating the transparent region withlight in the supplying step; (c) an optically analyzing means capable ofanalyzing a change in an optical intensity of the light; and (d) a meansfor determining a kind of the sample by the optical intensity.
 6. Theapparatus according to claim 5, wherein the supplying means is adispensing means in which a pipette tip can be mounted and liquid can beaspirated and poured out by means of the tip.
 7. The apparatus accordingto claim 5, wherein the supplying means is a tube or a channel.
 8. Theapparatus according to claim 5, wherein the sample is whole blood, aserum, or plasma.
 9. The method according to claim 2, wherein the sampleis whole blood, a serum, or plasma.
 10. The method according to claim 3,wherein the sample is whole blood, a serum, or plasma.
 11. The apparatusaccording to claim 6, wherein the sample is whole blood, a serum, orplasma.
 12. The apparatus according to claim 7, wherein the sample iswhole blood, a serum, or plasma.